The accident happened at Fukushima Daiichi Nuclear Power Station on Mar. 11, 2011 accompanying with Great East Japan Earthquake has brought increased public interests in radiation. Also, in Japan, cancer radiotherapies tend to increase in recent years, and more specific studies regarding a radiation-protecting agent that protects normal tissues from radiation, and a radiation-sensitizing agent intended for synergistically improving the therapeutic effect of cancer are demanded.
Many studies intended to protect tissues from injury by radiation have been made heretofore. At present, drugs that prevent radionuclides from being absorbed and depositing in a living body and urge discharge of the same, such as an iodine agent and a chelating agent are known. However, these are used exclusively for reducing massive internal exposure by a specific radioactive element, and efficacy for low internal exposure or external exposure that can occur in a medical treatment is not observed. As famous examples of a drug intended to protect normal tissues in radiotherapy of tumors, cysteamine and amifostine are known. However, these drugs have difficulty in common use because of the side effect or the like. Also, many precedent studies on a radiation-sensitizing agent have been performed, but there is no radiation-sensitizing agent that is actually applied in clinical sites.
Under such circumstances, it is urgently demanded to develop effective radiation protecting and mitigating agents, and radiation-sensitizing agents. For rapidly developing such drugs, it is necessary to rapidly screen candidate drugs.
Here, various methods have been proposed for screening influence of an object to be investigated on human beings or animals such as influence by irradiation with radiation, effect of a drug, toxicity of a drug and so on. Screening methods that have been already proposed will be described below.
<Conventional Method 1: Screening Method Based on Survival Rate of Experimental Animal Such as Mouse or Rabbit>
This method is a method for screening influence of an object to be investigated on an experimental animal including subjecting an experimental animal such as mouse or rabbit to irradiation with radiation, drug administration or the like, and then measuring the survival rate of the experimental animal (see Non-Patent Document 1).
This method has the following problems. That is, it requires 20 days to 30 days of experimental period. Since the number of samples examinable at a time is limited to about one sample, and several tens of animals are required for examination of one sample, the method requires much cost. A large quantity of drug, which is equal to or more than about 10 times the concentration in an in vitro experiment, is required in administering an experimental animal with the drug. Therefore, it is difficult to prepare samples, particularly, in early stages of new drug development. It is largely affected by individual differences of experimental animals.
<Conventional Method 2: Comet Assay Method>
This method is an assay of detecting a DNA fragment occurring by irradiation with radiation, drug administration or the like as a length of a tail that looks like a comet (see Non-Patent Document 2). The basic principle is as follows. That is, DNA is caused to migrate in an agarose gel by electrophoresis, and observed under a microscope. The cell having experienced a cut among the migration cells looks like a comet. This migration cell consists of a head part composed of a nuclear region, and a tail part composed of a DNA strand that is loosened by the cut and migrates to the anode. Variation in DNA migration amount depends on various factors including the agarose concentration of the gel, pH, temperature, and voltage, current and running time in the electrophoresis. This method is also employed as a method for identifying a toxic substance in toxicity evaluation assays of foods and chemicals, and researches on health effects.
This method has the following problems. That is, it requires 2 days to 3 days of experimental period. Since the method includes a large number of manipulation steps and requires time for the manipulation, the number of samples that can be examined at a time is about ten at most. It is costly because dedicated gels and reagents are required.
<Conventional Method 3: PCC Method>
This PCC (Premature chromosome condensation) method is a method of counting the number of chromosomes by observation under a microscope to identify injury of chromosomes qualitatively and quantitatively, in estimation of an exposure dose of an individual (see Non-Patent Documents 3, 4).
This method has the following problems. That is, it requires about one week of experimental period. Since the process requires time, the number of samples that can be examined at a time is about ten at most. It includes many manipulation steps. Visual check under a microscope by man power leads fatigue in data analysis. Visual check under a microscope by man power tends to involve a subjective view.
<Conventional Method 4: Multicolor FISH Method>
By combining the aforementioned PCC method (Conventional method 3) and a Multicolor FISH (Fluorescence in situ hybridization) method, it is possible to observe the details of injury of chromosome. Here, the Multicolor FISH method is a method for discriminating all chromosomes in a single hybridization manipulation. Probes that will specifically bind with respective chromosomes are provided, and each probe is labeled with a combination of five colors of fluorescent dyes so that the combination of fluorescent wavelengths differs between chromosomes. Each chromosome is discriminated according to the combination.
This method has the following problems. That is, it requires about one week of experimental period, and takes a time until the result is obtained. The number of samples that can be examined at a time is about one at most. Visual check under a microscope by man power leads fatigue in data analysis. Visual check under a microscope by man power tends to involve a subjective view. It is costly because it requires expensive probes or the like.
<Conventional Method 5: MTT Assay Method>
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) is used in checking influence and toxicity on cell growth. MTT, which is water-soluble and forms a yellow solution, turns into formazan by a dehydrogenase existing in mitochondria in cells after it is incorporated into the cells, and deposits as non-aqueous blue crystals. This deposit turns into a reddish violet solution when it is solved in an organic solvent such as DMSO (dimethyl sulfoxide), and thus the growth rate and survival rate of the cells can be determined as a mitochondria enzyme activity by absorbance at 450 nm. For example, in the case of HeLa cells (cells derived from human cervical cancer) having a doubling time of 24 hours, a MTT treatment is performed at a fixed time everyday (every 24 hours), and a growth curve of the cells can be determined from the measurements by a plate reader.
This method has the following problems. That is, it requires 3 days to 14 days of experimental period, and takes a time until the result is obtained. Also, much cost is required for reagents.
<Conventional Method 6: Trypan Blue Staining Method>
In this method, cells are stained with Trypan Blue (Trypan Blue tetrasodium; 3,3′-[(3,3′-dimethyl-4,4′-biphenylene)bis(azo)]bis[5-amino-4-hydroxy-2,7-naphthalene diphosphonate] dye, and apoptotic cells are counted with a hemocytometer (Neubauer hemocytometer) to detect cytotoxicity. Trypan Blue passes through a cell membrane having injury and stains an apoptotic cell blue, but it does not stain a normal cell having a perfect membrane. By counting the cells stained blue, it is possible to quantify the apoptosis.
This method has the following problems. That is, it requires 3 days to 7 days of experimental period, and takes a time until the result is obtained. The number of samples that can be examined at a time is about three at most. Visual check under a microscope by man power leads fatigue in data analysis. Visual check under a microscope by man power tends to involve a subjective view.
<Other Proposed Conventional Method>
Also, a method of screening a molecule that reduces apoptosis of cells is proposed (see Patent Document 1). In this method, Mcl-1 null MEF cells are seeded onto a flat-bottomed 96-well plate, and after 12 hours to 24 hours, a library compound is added at final concentrations of 0.1 μM, 1 μM, and 10 μM, and incubated for 2 hours, followed by addition of ABT-737 (100 nM) or a carrier medium, and cell viability is scored 24 hours later by using AlamarBlue dye and read 4 hours later.
This method has the following problems. That is, it requires at least about 2 days of experimental period, and takes a time until the result is obtained. Since the AlamarBlue dye or the like is used, time and labor are required for the manipulation.
As described above, any conventional method has at least one of the problems of taking a time for obtaining a result, requiring much cost, and susceptibility to a subjective view. There has been no method that can solve all of these three problems.